Generation of plants with improved pathogen resistance

ABSTRACT

The present invention is directed to plants that display a pathogen resistance phenotype due to altered expression of a PPR2 nucleic acid. The invention is further directed to methods of generating plants with a pathogen resistance phenotype.

BACKGROUND OF THE INVENTION

The control of infection by plant pathogens, which can inhibitproduction of fruits, seeds, foliage and flowers and cause reductions inthe quality and quantity of the harvested crops, is of significanteconomic importance. Pathogens annually cause billions of dollars indamage to crops worldwide (Baker et al. 1997, Science 276:726-733).Consequently, an increasing amount of research has been dedicated todeveloping novel methods for controlling plant diseases. Such studieshave centered on the plant's innate ability to resist pathogen invasionin an effort to buttress the plant's own defenses to counter pathogenattacks (Staskawicz et al. 1995, Science 268:661-667; Baker et al.supra).

Although most crops are treated with agricultural anti-fungal,anti-bacterial agents and/or pesticidal agents, damage from pathogenicinfection still results in revenue losses to the agricultural industryon a regular basis. Furthermore, many of the agents used to control suchinfection or infestation cause adverse side effects to the plant and/orto the environment. Plants with enhanced resistance to infection bypathogens would decrease or eliminate the need for application ofchemical anti-fungal, anti-bacterial and/or pesticidal agents.

There has been significant interest in developing transgenic plants thatshow increased resistance to a broad range of pathogens (Stuiver andCusters, 2001, Nature 411:865-8; Melchers and Stuiver, 2000, Curr OpinPlant Biol 3:147-52; Rommens and Kishore, 2000, Cuff Opin Biotechnol11:120-5; Mourgues et al. 1998, Trends Biotechnol 16:203-10). Theinteraction between Arabidopsis and the oomycete Peronospora parasitica(downy mildew) provides an attractive model system to identify molecularcomponents of the host that are required for recognition of the fungalparasite (Parker et al. 1996 Plant Cell 8:2033-46). A number of geneswhose mis-expression is associated with altered resistance to P.parasitica, as well as other pathogens, have been identified inArabidopsis. Overexpression of the NPR1 gene confers resistance toinfection by P. parasitica as well as the bacterial pathogen Pseudomonassyringae (Cao et al, 1998 Proc Natl Acad Sci USA 95:6531-6536). CPRE issemi-dominant mutation implicated in multiple defense pathways (Clarkeet al. 1998, Plant Cell 10:557-569). Lsd6 and Lsd7 are dominantmutations that confer heightened disease and result in the developmentof spontaneous necrotic lesions and elevated levels of salicylic acid(Weymann et al 1995 Plant Cell 7:2013-2022). A number of recessivemutations confer P. parasitica resistance, including ssi2, in the SSI2gene encoding a stearoyl-ACP desaturase (Kachroo et al. 2001 Proc NatlAcad Sci USA 98:9448-9453), mpk4, in a MAP kinase gene (Petersen et al.2000, Cell 103:1111-20), and pmr4 (Vogel and Somerville 2000 Proc NatlAcad Sci USA 97:1897-1902). The recessive mutations cpr5 and cpr1 alsoconfer resistance to P. syringae and cause a dwarf phenotype (Bowling etal 1997 Plant Cell 9:1573-1584; Bowling et al, 1994 Plant Cell6:1845-1857).

Activation tagging in plants refers to a method of generating randommutations by insertion of a heterologous nucleic acid constructcomprising regulatory sequences (e.g., an enhancer) into a plant genome.The regulatory sequences can act to enhance transcription of one or morenative plant genes; accordingly, activation tagging is a fruitful methodfor generating gain-of-function, generally dominant mutants (see, e.g.,Hayashi et al., Science (1992) 258: 1350-1353; Weigel et al., PlantPhysiology (2000) 122:1003-1013). The inserted construct provides amolecular tag for rapid identification of the native plant whosemis-expression causes the mutant phenotype. Activation tagging may alsocause loss-of-function phenotypes. The insertion may result indisruption of a native plant gene, in which case the phenotype isgenerally recessive.

Activation tagging has been used in various species, including tobaccoand Arabidopsis, to identify many different kinds of mutant phenotypesand the genes associated with these phenotypes (Wilson et al., PlantCell (1996) 8:659-671, Schaffer et al., Cell (1998) 93: 1219-1229;Fridborg et al., Plant Cell (1999)11: 1019-1032; Kardailsky et al.,Science (1999) 286:1962-1965); Christensen S et al., 9^(th)International Conference on Arabidopsis Research. Univ. ofWisconsin-Madison, Jun. 24-28, 1998. Abstract 165). In one example,activation tagging was used to identify mutants with altered diseaseresistance (Weigel et al., supra).

SUMMARY OF THE INVENTION

The invention provides a transgenic plant comprising a planttransformation vector comprising a nucleotide sequence that encodes oris complementary to a sequence that encodes a PPR2 polypeptide or anortholog thereof. The transgenic plant is characterized by havingincreased resistance to pathogens.

The present invention further provides a method of producing an alteredpathogen resistance phenotype in a plant. The method comprisesintroducing into plant progenitor cells a vector comprising a nucleotidesequence that encodes or is complementary to a sequence encoding a PPR2polypeptide or an ortholog thereof and growing a transgenic plant thatexpresses the nucleotide sequence. In one embodiment, the PPR2polypeptide has at least 50% sequence identity to the amino acidsequence presented in SEQ ID NO:2 and comprises a SANT domain. In otherembodiments, the PPR2 polypeptide has at least 80% or 90% sequenceidentity to or has the amino acid sequence presented in SEQ ID NO:2.

The invention further provides plants and plant parts obtained by themethods described herein.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless otherwise indicated, all technical and scientific terms usedherein have the same meaning as they would to one skilled in the art ofthe present invention. Practitioners are particularly directed toSambrook et al., Molecular Cloning: A Laboratory Manual (SecondEdition), Cold Spring Harbor Press, Plainview, N.Y., 1989, and Ausubel FM et al., Current Protocols in Molecular Biology, John Wiley & Sons, NewYork, N.Y., 1993, for definitions and terms of the art. It is to beunderstood that this invention is not limited to the particularmethodology, protocols, and reagents described, as these may vary.

As used herein, the term “vector” refers to a nucleic acid constructdesigned for transfer between different host cells. An “expressionvector” refers to a vector that has the ability to incorporate andexpress heterologous DNA fragments in a foreign cell. Many prokaryoticand eukaryotic expression vectors are commercially available. Selectionof appropriate expression vectors is within the knowledge of thosehaving skill in the art.

A “heterologous” nucleic acid construct or sequence has a portion of thesequence that is not native to the plant cell in which it is expressed.Heterologous, with respect to a control sequence refers to a controlsequence (i.e. promoter or enhancer) that does not function in nature toregulate the same gene the expression of which it is currentlyregulating. Generally, heterologous nucleic acid sequences are notendogenous to the cell or part of the genome in which they are present,and have been added to the cell, by infection, transfection,microinjection, electroporation, or the like. A “heterologous” nucleicacid construct may contain a control sequence/DNA coding sequencecombination that is the same as, or different from a controlsequence/DNA coding sequence combination found in the native plant.

As used herein, the term “gene” means the segment of DNA involved inproducing a polypeptide chain, which may or may not include regionspreceding and following the coding region, e.g. 5′ untranslated (5′ UTR)or “leader” sequences and 3′ UTR or “trailer” sequences, as well asintervening sequences (introns) between individual coding segments(exons) and non-transcribed regulatory sequence.

As used herein, “recombinant” includes reference to a cell or vector,that has been modified by the introduction of a heterologous nucleicacid sequence or that the cell is derived from a cell so modified. Thus,for example, recombinant cells express genes that are not found inidentical form within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all as a result of deliberate humanintervention.

As used herein, the term “gene expression” refers to the process bywhich a polypeptide is produced based on the nucleic acid sequence of agene. The process includes both transcription and translation;accordingly, “expression” may refer to either a polynucleotide orpolypeptide sequence, or both. Sometimes, expression of a polynucleotidesequence will not lead to protein translation. “Over-expression” refersto increased expression of a polynucleotide and/or polypeptide sequencerelative to its expression in a wild-type (or other reference [e.g.,non-transgenic]) plant and may relate to a naturally-occurring ornon-naturally occurring sequence. “Ectopic expression” refers toexpression at a time, place, and/or increased level that does notnaturally occur in the non-altered or wild-type plant.“Under-expression” refers to decreased expression of a polynucleotideand/or polypeptide sequence, generally of an endogenous gene, relativeto its expression in a wild-type plant. The terms “mis-expression” and“altered expression” encompass over-expression, under-expression, andectopic expression.

The term “introduced” in the context of inserting a nucleic acidsequence into a cell, means “transfection”, or “transformation” or“transduction” and includes reference to the incorporation of a nucleicacid sequence into a eukaryotic or prokaryotic cell where the nucleicacid sequence may be incorporated into the genome of the cell (forexample, chromosome, plasmid, plastid, or mitochondrial DNA), convertedinto an autonomous replicon, or transiently expressed (for example,transfected mRNA).

As used herein, a “plant cell” refers to any cell derived from a plant,including cells from undifferentiated tissue (e.g., callus) as well asplant seeds, pollen, progagules and embryos.

As used herein, the terms “native” and “wild-type” relative to a givenplant trait or phenotype refers to the form in which that trait orphenotype is found in the same variety of plant in nature.

As used herein, the term “modified” regarding a plant trait, refers to achange in the phenotype of a transgenic plant relative to the similarnon-transgenic plant. An “interesting phenotype (trait)” with referenceto a transgenic plant refers to an observable or measurable phenotypedemonstrated by a T1 and/or subsequent generation plant, which is notdisplayed by the corresponding non-transgenic (i.e., a genotypicallysimilar plant that has been raised or assayed under similar conditions).An interesting phenotype may represent an improvement in the plant ormay provide a means to produce improvements in other plants. An“improvement” is a feature that may enhance the utility of a plantspecies or variety by providing the plant with a unique and/or novelquality.

An “altered pathogen resistance phenotype” refers to detectable changein the response of a genetically modified plant to pathogenic infection,compared to the similar, but non-modified plant. The phenotype may beapparent in the plant itself (e.g., in growth, viability or particulartissue morphology of the plant) or may be apparent in the ability of thepathogen to proliferate on and/or infect the plant. As used herein,“improved pathogen resistance” refers to increased resistance to apathogen.

As used herein, a “mutant” polynucleotide sequence or gene differs fromthe corresponding wild type polynucleotide sequence or gene either interms of sequence or expression, where the difference contributes to amodified plant phenotype or trait. Relative to a plant or plant line,the term “mutant” refers to a plant or plant line which has a modifiedplant phenotype or trait, where the modified phenotype or trait isassociated with the modified expression of a wild type polynucleotidesequence or gene.

As used herein, the term “T1” refers to the generation of plants fromthe seed of T0 plants. The T1 generation is the first set of transformedplants that can be selected by application of a selection agent, e.g.,an antibiotic or herbicide, for which the transgenic plant contains thecorresponding resistance gene. The term “T2” refers to the generation ofplants by self-fertilization of the flowers of T1 plants, previouslyselected as being transgenic.

As used herein, the term “plant part” includes any plant organ ortissue, including, without limitation, seeds, embryos, meristematicregions, callus tissue, leaves, roots, shoots, gametophytes,sporophytes, pollen, and microspores. Plant cells can be obtained fromany plant organ or tissue and cultures prepared therefrom. The class ofplants which can be used in the methods of the present invention isgenerally as broad as the class of higher plants amenable totransformation techniques, including both monocotyledenous anddicotyledenous plants.

As used herein, “transgenic plant” includes reference to a plant thatcomprises within its genome a heterologous polynucleotide. Theheterologous polynucleotide can be either stably integrated into thegenome, or can be extra-chromosomal. Preferably, the polynucleotide ofthe present invention is stably integrated into the genome such that thepolynucleotide is passed on to successive generations. A plant cell,tissue, organ, or plant into which the heterologous polynucleotides havebeen introduced is considered “transformed”, “transfected”, or“transgenic”. Direct and indirect progeny of transformed plants or plantcells that also contain the heterologous polynucleotide are alsoconsidered transgenic.

Identification of Plants with an Improved Pathogen Resistance Phenotype

We used an Arabidopsis activation tagging screen to identify theassociation between the gene we have designated “PPR2 (for P. garasiticaResistant),” predicted to encode a myb-related protein, and an alteredpathogen resistance phenotype, specifically, increased resistance to thefungal pathogen P. parasitica (downy mildew). Briefly, and as furtherdescribed in the Examples, a large number of Arabidopsis plants weremutated with the pSKI015 vector, which comprises a T-DNA from the Tiplasmid of Agrobacterium tumifaciens, a viral enhancer element, and aselectable marker gene (Weigel et al, supra). When the T-DNA insertsinto the genome of transformed plants, the enhancer element can causeup-regulation genes in the vicinity, generally within about 10 kilobase(kb) of the insertion. T1 plants were exposed to the selective agent inorder to specifically recover transformed plants that expressed theselectable marker and therefore harbored T-DNA insertions. Samples ofapproximately 18 T2 seed were planted, grown to seedlings, andinoculated with P. parasitica spores. Disease symptoms on individualplants were scored based on the number of conidiophores that emerged.Accordingly, plants on which growth of conidiophores was reduced wereidentified as pathogen resistant.

An Arabidopsis line that showed increased resistance to P. parasiticainfection was identified. The association of the PPR2 gene with thepathogen resistance phenotype was discovered by analysis of the genomicDNA sequence flanking the T-DNA insertion in the identified line.Accordingly, PPR2 genes and/or polypeptides may be employed in thedevelopment of genetically modified plants having a modified pathogenresistance phenotype. PPR2 genes may be used in the generation of cropsand/or other plant species that have improved resistance to infection byP. parasitica and other oomycetes and may also be useful the generationof plant with improved resistance to fungal, bacterial, and/or otherpathogens. Mis-expression of PPR2 genes may thus reduce the need forfungicides and/or pesticides. The modified pathogen resistance phenotypemay further enhance the overall health of the plant.

PPR2 Nucleic Acids and Polypeptides

Arabidopsis PPR2 nucleic acid (coding) sequence is provided in SEQ IDNO:1 and in Genbank entry GI 12331602, nucleotides 20955-21335(designated F22H5.3 and At1g75250). The corresponding protein sequenceis provided in SEQ ID NO:2 and in GI 10092271.

As used herein, the term “PPR2 polypeptide” refers to a full-length PPR2protein or a fragment, derivative (variant), or ortholog thereof that is“functionally active,” meaning that the protein fragment, derivative, orortholog exhibits one or more or the functional activities associatedwith the polypeptide of SEQ ID NO:2. In one preferred embodiment, afunctionally active PPR2 polypeptide causes an altered pathogenresistance phenotype when mis-expressed in a plant. In a furtherpreferred embodiment, mis-expression of the functionally active PPR2polypeptide causes increased resistance to P. parasitica and/or otheroomycetes. In another embodiment, a functionally active PPR2 polypeptideis capable of rescuing defective (including deficient) endogenous PPR2activity when expressed in a plant or in plant cells; the rescuingpolypeptide may be from the same or from a different species as thatwith defective activity. In another embodiment, a functionally activefragment of a full length PPR2 polypeptide (i.e., a native polypeptidehaving the sequence of SEQ ID NO:2 or a naturally occurring orthologthereof) retains one of more of the biological properties associatedwith the full-length PPR2 polypeptide, such as signaling activity,binding activity, catalytic activity, or cellular or extra-cellularlocalizing activity. Some preferred PPR2 polypeptides display DNAbinding activity. A PPR2 fragment preferably comprises a PPR2 domain,such as a C- or N-terminal or catalytic domain, among others, andpreferably comprises at least 10, preferably at least 20, morepreferably at least 25, and most preferably at least 50 contiguous aminoacids of a PPR2 protein. Functional domains can be identified using thePFAM program (Bateman A et al., 1999 Nucleic Acids Res 27:260-262;website at pfam.wustl.edu). A preferred PPR2 fragment comprises a SANTdomain (SM00395) identified by PFAM, at approximately amino acids 8-60.Functionally active variants of full-length PPR2 polypeptides orfragments thereof include polypeptides with amino acid insertions,deletions, or substitutions that retain one of more of the biologicalproperties associated with the full-length PPR2 polypeptide. In somecases, variants are generated that change the post-translationalprocessing of a PPR2 polypeptide. For instance, variants may havealtered protein transport or protein localization characteristics oraltered protein half-life compared to the native polypeptide.

As used herein, the term “PPR2 nucleic acid” encompasses nucleic acidswith the sequence provided in or complementary to the sequence providedin SEQ ID NO:1, as well as functionally active fragments, derivatives,or orthologs thereof. A PPR2 nucleic acid of this invention may be DNA,derived from genomic DNA or cDNA, or RNA.

In one embodiment, a functionally active PPR2 nucleic acid encodes or iscomplementary to a nucleic acid that encodes a functionally active PPR2polypeptide. Included within this definition is genomic DNA that servesas a template for a primary RNA transcript (i.e., an mRNA precursor)that requires processing, such as splicing, before encoding thefunctionally active PPR2 polypeptide. A PPR2 nucleic acid can includeother non-coding sequences, which may or may not be transcribed; suchsequences include 5′ and 3′ UTRs, polyadenylation signals and regulatorysequences that control gene expression, among others, as are known inthe art. Some polypeptides require processing events, such asproteolytic cleavage, covalent modification, etc., in order to becomefully active. Accordingly, functionally active nucleic acids may encodethe mature or the pre-processed PPR2 polypeptide, or an intermediateform. A PPR2 polynucleotide can also include heterologous codingsequences, for example, sequences that encode a marker included tofacilitate the purification of the fused polypeptide, or atransformation marker.

In another embodiment, a functionally active PPR2 nucleic acid iscapable of being used in the generation of loss-of-function pathogenresistance phenotypes, for instance, via antisense suppression,co-suppression, etc.

In one preferred embodiment, a PPR2 nucleic acid used in the methods ofthis invention comprises a nucleic acid sequence that encodes or iscomplementary to a sequence that encodes a PPR2 polypeptide having atleast 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identityto the polypeptide sequence presented in SEQ ID NO:2.

In another embodiment a PPR2 polypeptide of the invention comprises apolypeptide sequence with at least 50% or 60% identity to the PPR2polypeptide sequence of SEQ ID NO:2, and may have at least 70%, 80%,85%, 90% or 95% or more sequence identity to the PPR2 polypeptidesequence of SEQ ID NO:2. In another embodiment, a PPR2 polypeptidecomprises a polypeptide sequence with at least 50%, 60%, 70%, 80%, 85%,90% or 95% or more sequence identity to a functionally active fragmentof the polypeptide presented in SEQ ID NO:2, such as a SANT domain. Inyet another embodiment, a PPR2 polypeptide comprises a polypeptidesequence with at least 50%, 60%, 70%, 80%, or 90% identity to thepolypeptide sequence of SEQ ID NO:2 over its entire length and comprisesa SANT domain.

In another aspect, a PPR2 polynucleotide sequence is at least 50% to 60%identical over its entire length to the PPR2 nucleic acid sequencepresented as SEQ ID NO:1, or nucleic acid sequences that arecomplementary to such a PPR2 sequence, and may comprise at least 70%,80%, 85%, 90% or 95% or more sequence identity to the PPR2 sequencepresented as SEQ ID NO:1 or a functionally active fragment thereof, orcomplementary sequences.

As used herein, “percent (%) sequence identity” with respect to aspecified subject sequence, or a specified portion thereof, is definedas the percentage of nucleotides or amino acids in the candidatederivative sequence identical with the nucleotides or amino acids in thesubject sequence (or specified portion thereof), after aligning thesequences and introducing gaps, if necessary to achieve the maximumpercent sequence identity, as generated by the program WU-BLAST-2.0a19(Altschul et al., J. Mol. Biol. (1990) 215:403-410; website atblastwustl.edu/blast/README.html) with search parameters set to defaultvalues. The HSP S and HSP S2 parameters are dynamic values and areestablished by the program itself depending upon the composition of theparticular sequence and composition of the particular database againstwhich the sequence of interest is being searched. A “% identity value”is determined by the number of matching identical nucleotides or aminoacids divided by the sequence length for which the percent identity isbeing reported. “Percent (%) amino acid sequence similarity” isdetermined by doing the same calculation as for determining % amino acidsequence identity, but including conservative amino acid substitutionsin addition to identical amino acids in the computation. A conservativeamino acid substitution is one in which an amino acid is substituted foranother amino acid having similar properties such that the folding oractivity of the protein is not significantly affected. Aromatic aminoacids that can be substituted for each other are phenylalanine,tryptophan, and tyrosine; interchangeable hydrophobic amino acids areleucine, isoleucine, methionine, and valine; interchangeable polar aminoacids are glutamine and asparagine; interchangeable basic amino acidsare arginine, lysine and histidine; interchangeable acidic amino acidsare aspartic acid and glutamic acid; and interchangeable small aminoacids are alanine, serine, threonine, cysteine and glycine.

Derivative nucleic acid molecules of the subject nucleic acid moleculesinclude sequences that hybridize to the nucleic acid sequence of SEQ IDNO:1. The stringency of hybridization can be controlled by temperature,ionic strength, pH, and the presence of denaturing agents such asformamide during hybridization and washing. Conditions routinely usedare well known (see, e.g., Current Protocol in Molecular Biology, Vol.1, Chap. 2.10, John Wiley & Sons, Publishers (1994); Sambrook et al.,supra). In some embodiments, a nucleic acid molecule of the invention iscapable of hybridizing to a nucleic acid molecule containing thenucleotide sequence of SEQ ID NO:1 under stringent hybridizationconditions that comprise: prehybridization of filters containing nucleicacid for 8 hours to overnight at 65° C. in a solution comprising 6×single strength citrate (SSC) (1×SSC is 0.15 M NaCl, 0.015 M Na citrate;pH 7.0), 5× Denhardt's solution, 0.05% sodium pyrophosphate and 100μg/ml herring sperm DNA; hybridization for 18-20 hours at 65° C. in asolution containing 6×SSC, 1× Denhardt's solution, 100 μg/ml yeast tRNAand 0.05% sodium pyrophosphate; and washing of filters at 65° C. for 1 hin a solution containing 0.2×SSC and 0.1% SDS (sodium dodecyl sulfate).In other embodiments, moderately stringent hybridization conditions areused that comprise: pretreatment of filters containing nucleic acid for6 h at 40° C. in a solution containing 35% formamide, 5×SSC, 50 mMTris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500μg/ml denatured salmon sperm DNA; hybridization for 18-20 h at 40° C. ina solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA,and 10% (wt/vol) dextran sulfate; followed by washing twice for 1 hourat 55° C. in a solution containing 2×SSC and 0.1% SDS. Alternatively,low stringency conditions can be used that comprise: incubation for 8hours to overnight at 37° C. in a solution comprising 20% formamide,5×SSC, 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10%dextran sulfate, and 20 μg/ml denatured sheared salmon sperm DNA;hybridization in the same buffer for 18 to 20 hours; and washing offilters in 1×SSC at about 37° C. for 1 hour.

As a result of the degeneracy of the genetic code, a number ofpolynucleotide sequences encoding a PPR2 polypeptide can be produced.For example, codons may be selected to increase the rate at whichexpression of the polypeptide occurs in a particular host species, inaccordance with the optimum codon usage dictated by the particular hostorganism (see, e.g., Nakamura Y et al, Nucleic Acids Res (1999) 27:292).Such sequence variants may be used in the methods of this invention.

The methods of the invention may use orthologs of the Arabidopsis PPR2.Methods of identifying the orthologs in other plant species are known inthe art. Normally, orthologs in different species retain the samefunction, due to presence of one or more protein motifs and/or3-dimensional structures. In evolution, when a gene duplication eventfollows speciation, a single gene in one species, such as Arabidopsis,may correspond to multiple genes (paralogs) in another. As used herein,the term “orthologs” encompasses paralogs. When sequence data isavailable for a particular plant species, orthologs are generallyidentified by sequence homology analysis, such as BLAST analysis,usually using protein bait sequences. Sequences are assigned as apotential ortholog if the best hit sequence from the forward BLASTresult retrieves the original query sequence in the reverse BLAST(Huynen M A and Bork P, Proc Natl Acad Sci (1998) 95:5849-5856; Huynen MA et al., Genome Research (2000) 10:1204-1210). Programs for multiplesequence alignment, such as CLUSTAL (Thompson J D et al, 1994, NucleicAcids Res 22:4673-4680) may be used to highlight conserved regionsand/or residues of orthologous proteins and to generate phylogenetictrees. In a phylogenetic tree representing multiple homologous sequencesfrom diverse species (e.g., retrieved through BLAST analysis),orthologous sequences from two species generally appear closest on thetree with respect to all other sequences from these two species.Structural threading or other analysis of protein folding (e.g., usingsoftware by ProCeryon, Biosciences, Salzburg, Austria) may also identifypotential orthologs. Nucleic acid hybridization methods may also be usedto find orthologous genes and are preferred when sequence data are notavailable. Degenerate PCR and screening of cDNA or genomic DNA librariesare common methods for finding related gene sequences and are well knownin the art (see, e.g., Sambrook, supra; Dieffenbach and Dveksler (Eds.)PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press,NY, 1989). For instance, methods for generating a cDNA library from theplant species of interest and probing the library with partiallyhomologous gene probes are described in Sambrook et al. A highlyconserved portion of the Arabidopsis PPR2 coding sequence may be used asa probe. PPR2 ortholog nucleic acids may hybridize to the nucleic acidof SEQ ID NO:1 under high, moderate, or low stringency conditions. Afteramplification or isolation of a segment of a putative ortholog, thatsegment may be cloned and sequenced by standard techniques and utilizedas a probe to isolate a complete cDNA or genomic clone. Alternatively,it is possible to initiate an EST project to generate a database ofsequence information for the plant species of interest. In anotherapproach, antibodies that specifically bind known PPR2 polypeptides areused for ortholog isolation. Western blot analysis can determine that aPPR2 ortholog (i.e., an orthologous protein) is present in a crudeextract of a particular plant species. When reactivity is observed, thesequence encoding the candidate ortholog may be isolated by screeningexpression libraries representing the particular plant species.Expression libraries can be constructed in a variety of commerciallyavailable vectors, including lambda gt11, as described in Sambrook, etal., supra. Once the candidate ortholog(s) are identified by any ofthese means, candidate orthologous sequence are used as bait (the“query”) for the reverse BLAST against sequences from Arabidopsis orother species in which PPR2 nucleic acid and/or polypeptide sequenceshave been identified.

PPR2 nucleic acids and polypeptides may be obtained using any availablemethod. For instance, techniques for isolating cDNA or genomic DNAsequences of interest by screening DNA libraries or by using polymerasechain reaction (PCR), as previously described, are well known in theart. Alternatively, nucleic acid sequence may be synthesized. Any knownmethod, such as site directed mutagenesis (Kunkel T A et al., MethodsEnzymol. (1991) 204:125-39), may be used to introduce desired changesinto a cloned nucleic acid.

In general, the methods of the invention involve incorporating thedesired form of the PPR2 nucleic acid into a plant expression vector fortransformation of in plant cells, and the PPR2 polypeptide is expressedin the host plant.

An isolated PPR2 nucleic acid molecule is other than in the form orsetting in which it is found in nature and is identified and separatedfrom least one contaminant nucleic acid molecule with which it isordinarily associated in the natural source of the PPR2 nucleic acid.However, an isolated PPR2 nucleic acid molecule includes PPR2 nucleicacid molecules contained in cells that ordinarily express PPR2 where,for example, the nucleic acid molecule is in a chromosomal locationdifferent from that of natural cells.

Generation of Genetically Modified Plants with a Pathogen ResistancePhenotype

PPR2 nucleic acids and polypeptides may be used in the generation ofgenetically modified plants having a modified pathogen resistancephenotype; in general, improved resistance phenotypes are of interest.Pathogenic infection may affect seeds, fruits, blossoms, foliage, stems,tubers, roots, etc. Accordingly, resistance may be observed in any partof the plant. In a preferred embodiment, altered expression of the PPR2gene in a plant is used to generate plants with increased resistance toP. parasitica. In a further preferred embodiment, plants thatmis-express PPR2 may also display altered resistance to other pathogens.Other oomycete pathogens of interest include Pythium spp, Phytophthoraspp, Bremia lactucae, Peronosclerospora spp., Pseudoperonospora.Sclerophthora macrospora, Sclerospora graminicola, Plasmopara viticola,and Albugo candidia. Fungal pathogens of interest include Alternariabrassicicola, Botrytis cinerea, Erysiphe cichoracearum, Fusariumoxysporum, Plasmodiophora brassica, Rhizoctonia solani, Colletotrichumcoccode, Sclerotinia spp., Aspergillus spp., Penicillium spp., Ustilagospp., and Tilletia spp. Bacterial pathogens of interest includeAgrobacterium tumefaciens, Erwinia tracheiphila, Erwinia stewartii,Xanthomonas phaseoli, Erwinia amylovora, Erwinia carotovora, Pseudomonassyringae, Pelargonium spp, Pseudomonas cichorii, Xanthomonas fragariae,Pseudomonas morsprunorum, Xanthomonas campestris.

The methods described herein are generally applicable to all plants.Although activation tagging and gene idetitification is carried out inArabidopsis, the PPR2 gene (or an ortholog, variant or fragment thereof)may be expressed in any type of plant. In preferred embodiments, theinvention is directed to crops including maize, soybean, cotton, rice,wheat, barley, tomato, canola, turfgrass, and flax. Other crops includealfalfa, tobacco, and other forage crops. The invention may also bedirected to fruit- and vegetable-bearing plants, plants used in the cutflower industry, grain-producing plants, oil-producing plants, andnut-producing plants, among others.

The skilled artisan will recognize that a wide variety of transformationtechniques exist in the art, and new techniques are continually becomingavailable. Any technique that is suitable for the target host plant canbe employed within the scope of the present invention. For example, theconstructs can be introduced in a variety of forms including, but notlimited to as a strand of DNA, in a plasmid, or in an artificialchromosome. The introduction of the constructs into the target plantcells can be accomplished by a variety of techniques, including, but notlimited to Agrobacterium-mediated transformation, electroporation,microinjection, microprojectile bombardment calcium-phosphate-DNAco-precipitation or liposome-mediated transformation of a heterologousnucleic acid. The transformation of the plant is preferably permanent,i.e. by integration of the introduced expression constructs into thehost plant genome, so that the introduced constructs are passed ontosuccessive plant generations. Depending upon the intended use, aheterologous nucleic acid construct comprising a PPR2 polynucleotide mayencode the entire protein or a biologically active portion thereof.

In one embodiment, binary Ti-based vector systems may be used totransfer polynucleotides. Standard Agrobacterium binary vectors areknown to those of skill in the art, and many are commercially available(e.g., pBI121 Clontech Laboratories, Palo Alto, Calif.).

The optimal procedure for transformation of plants with Agrobacteriumvectors will vary with the type of plant being transformed. Exemplarymethods for Agrobacterium-mediated transformation include transformationof explants of hypocotyl, shoot tip, stem or leaf tissue, derived fromsterile seedlings and/or plantlets. Such transformed plants may bereproduced sexually, or by cell or tissue culture. Agrobacteriumtransformation has been previously described for a large number ofdifferent types of plants and methods for such transformation may befound in the scientific literature.

Expression (including transcription and translation) of PPR2 may beregulated with respect to the level of expression, the tissue type(s)where expression takes place and/or developmental stage of expression. Anumber of heterologous regulatory sequences (e.g., promoters andenhancers) are available for controlling the expression of a PPR2nucleic acid. These include constitutive, inducible and regulatablepromoters, as well as promoters and enhancers that control expression ina tissue- or temporal-specific manner. Exemplary constitutive promotersinclude the raspberry E4 promoter (U.S. Pat. Nos. 5,783,393 and5,783,394), the 35S CaMV (Jones J D et al, Transgenic Res (1992)1:285-297), the CsVMV promoter (Verdaguer B et al., Plant Mol Biol(1998) 37:1055-1067) and the melon actin promoter (published PCTapplication WO0056863). Exemplary tissue-specific promoters include thetomato E4 and E8 promoters (U.S. Pat. No. 5,859,330) and the tomato 2AIIgene promoter (Van Haaren M J J et al., Plant Mol Bio (1993)21:625-640). In one preferred embodiment, PPR2 expression is under thecontrol of a pathogen-inducible promoter (Rushton et al., The Plant Cell(2002) 14:749-762).

In one preferred embodiment, PPR2 expression is under control ofregulatory sequences from genes whose expression is associated with theCsVMV promoter.

In yet another aspect, in some cases it may be desirable to inhibit theexpression of endogenous PPR2 in a host cell. Exemplary methods forpracticing this aspect of the invention include, but are not limited toantisense suppression (Smith, et al., Nature (1988) 334:724-726; van derKrol et al., Biotechniques (1988) 6:958-976); co-suppression (Napoli, etal., Plant Cell (1990) 2:279-289); ribozymes (PCT Publication WO97/10328); and combinations of sense and antisense (Waterhouse, et al.,Proc. Natl. Mad. Sci. USA (1998) 95:13959-13964). Methods for thesuppression of endogenous sequences in a host cell typically employ thetranscription or transcription and translation of at least a portion ofthe sequence to be suppressed. Such sequences may be homologous tocoding as well as non-coding regions of the endogenous sequence.Antisense inhibition may use the entire cDNA sequence (Sheehy et al.,Proc. Natl. Acad. Sci. USA (1988) 85:8805-8809), a partial cDNA sequenceincluding fragments of 5′ coding sequence, (Cannon et al., Plant Molec.Biol. (1990) 15:39-47), or 3′ non-coding sequences (Ch'ng et al., Proc.Natl. Acad. Sci. USA (1989) 86:10006-10010). Cosuppression techniquesmay use the entire cDNA sequence (Napoli et al., supra; van der Krol etal., The Plant Cell (1990) 2:291-299), or a partial cDNA sequence (Smithet al., Mol. Gen. Genetics (1990) 224:477-481).

Standard molecular and genetic tests may be performed to further analyzethe association between a gene and an observed phenotype. Exemplarytechniques are described below.

1. DNA/RNA Analysis

The stage- and tissue-specific gene expression patterns in mutant versuswild-type lines may be determined, for instance, by in situhybridization. Analysis of the methylation status of the gene,especially flanking regulatory regions, may be performed. Other suitabletechniques include overexpression, ectopic expression, expression inother plant species and gene knock-out (reverse genetics, targetedknock-out, viral induced gene silencing [VIGS, see Baulcombe D, (1999)Arch Virol Suppl 15:189-201]).

In a preferred application expression profiling, generally by microarrayanalysis, is used to simultaneously measure differences or inducedchanges in the expression of many different genes. Techniques formicroarray analysis are well known in the art (Schena M et al., Science(1995) 270:467-470; Baldwin D et al., Cur Opin Plant Biol. (1999)2(2):96-103; Dangond F, Physiol Genomics (2000) 2:53-58; van Hal N L etal., J Biotechnol (2000) 78:271-280; Richmond T and Somerville S, CurrOpin Plant Biol (2000) 3:108-116). Expression profiling of individualtagged lines may be performed. Such analysis can identify other genesthat are coordinately regulated as a consequence of the overexpressionof the gene of interest, which may help to place an unknown gene in aparticular pathway.

2. Gene Product Analysis

Analysis of gene products may include recombinant protein expression,antisera production, immunolocalization, biochemical assays forcatalytic or other activity, analysis of phosphorylation status, andanalysis of interaction with other proteins via yeast two-hybrid assays.

3. Pathway Analysis

Pathway analysis may include placing a gene or gene product within aparticular biochemical, metabolic or signaling pathway based on itsmis-expression phenotype or by sequence homology with related genes.Alternatively, analysis may comprise genetic crosses with wild-typelines and other mutant lines (creating double mutants) to order the genein a pathway, or determining the effect of a mutation on expression ofdownstream “reporter” genes in a pathway.

Generation of Mutated Plants with a Pathogen Resistance Phenotype

The invention further provides a method of identifying plants that havemutations in endogenous PPR2 that confer increased pathogen resistance,and generating pathogen-resistant progeny of these plants that are notgenetically modified. In one method, called “TILLING” (for targetinginduced local lesions in genomes), mutations are induced in the seed ofa plant of interest, for example, using EMS treatment. The resultingplants are grown and self-fertilized, and the progeny are used toprepare DNA samples. PPR2-specific PCR are used to identify whether amutated plant has a PPR2 mutation. Plants having PPR2 mutations may thenbe tested for pathogen resistance, or alternatively, plants may betested for pathogen resistance, and then PPR2-specific PCR is used todetermine whether a plant having increased pathogen resistance has amutated PPR2 gene. TILLING can identify mutations that may alter theexpression of specific genes or the activity of proteins encoded bythese genes (see Colbert et al (2001) Plant Physiol 126:480-484;McCallum et al (2000) Nature Biotechnology 18:455-457).

In another method, a candidate gene/Quantitative Trait Locus (QTLs)approach can be used in a marker-assisted breeding program to identifyalleles of or mutations in the PPR2 gene or orthologs of PPR2 that mayconfer increased resistance to pathogens (see Foolad et al., Theor ApplGenet. (2002) 104(6-7):945-958; Rothan et al., Theor Appl Genet (2002)105(1):145-159); Dekkers and Hospital, Nat Rev Genet. (2002) January;3(1):22-32). Thus, in a further aspect of the invention, a PPR2 nucleicacid is used to identify whether a plant having increased pathogenresistance has a mutation in endogenous PPR2 or has a particular allelethat causes the increased pathogen resistance.

While the invention has been described with reference to specificmethods and embodiments, it will be appreciated that variousmodifications and changes may be made without departing from theinvention. All publications cited herein are expressly incorporatedherein by reference for the purpose of describing and disclosingcompositions and methodologies that might be used in connection with theinvention. All cited patents, patent applications, and sequenceinformation in referenced websites and public databases are alsoincorporated by reference.

EXAMPLES Example 1 Generation of Plants with a PPR2 Phenotype byTransformation with an Activation Tagging Construct

Mutants were generated using the activation tagging “ACTTAG” vector,pSKI015 (GI 6537289; Weigel D et al., 2000). Standard methods were usedfor the generation of Arabidopsis transgenic plants, and wereessentially as described in published application PCT WO0183697.Briefly, T0 Arabidopsis (Col-0) plants were transformed withAgrobacterium carrying the pSKI015 vector, which comprises T-DNA derivedfrom the Agrobacterium Ti plasmid, an herbicide resistance selectablemarker gene, and the 4×CaMV 35S enhancer element. Transgenic plants wereselected at the T1 generation based on herbicide resistance. T2 seed wascollected from T1 plants and stored in an indexed collection, and aportion of the T2 seed was accessed for the screen.

Approximately 18 T2 seeds from each of the greater than 40,00 linestested were planted in soil. The seed were stratified for three days andthen grown in the greenhouse for seven days. The seedlings wereinoculated with approximately 1×10⁵ conidia per ml P. parasitica sporesand incubated in a dew room at 18° C. and 100% humidity for 24 hours.The plants were then moved to a growth room at 20° C. and 60% relativehumidity with ten-hour long light period for six days. Individual plantswere evaluated for the presence or absence of conidiophores oncotyledons. Lines in which at least a single plant showed noconidiophore growth were re-tested in a secondary screen by releasingthree sets of 18 seed and screening for resistance to P. parasiticagrowth as before.

Lines in which a significant number of plants showed no conidiophoresafter infection were subjected to a tertiary screen. Approximately 54 T2seed were released, planted individually and infected with P. parasiticaas before. The plants were evaluated for the number of conidiophoresgrowing on a single cotyledon and ranked by the following scoringsystem: a score of 0 indicates 0 conidiophores per cotyledon, 1indicates 1-5 conidiophores per cotyledon, 2 indicates 6-10conidiophores per cotyledon, 3 indicates 11-20 conidiophores percotyledon, and 4 indicates greater than 20 conidiophores per cotyledon

The ACTTAG line designated W000058335 was identified as having anincreased resistance phenotype. Specifically, 15.2% of individual plantsshowed no conidiophores in the secondary screen. In the tertiary screen,31 plants scored as 0 (39.2%), 31 as 1 (39.2%), 4 as 2 (5.1%), 10 as 3(12.7%)and 3 as 4 (3.8%). Control wild-type Col-0 plants were moresusceptible; 36 plants scored 0 (7.6%), 21 as 1 (4.4%), 79 as 2 (16.6%),250 as 3 (52.5%) and 90 as 4 (18.9%).

Example 2 Characterization of the T-DNA Insertion in Plants Exhibitingthe Altered Pathogen Resistance Phenotype

We performed standard molecular analyses, essentially as described inpatent application PCT WO0183697, to determine the site of the T-DNAinsertion associated with the increased pathogen resistance phenotype.Briefly, genomic DNA was extracted from plants exhibiting increasedpathogen resistance. PCR, using primers specific to the pSKI015 vector,confirmed the presence of the 35S enhancer in plants from lineW000058335, and Southern blot analysis verified the genomic integrationof the ACTTAG T-DNA and showed the presence of a single T-DNA insertionin the transgenic line.

Plasmid rescue and inverse PCR were used to recover genomic DNA flankingthe T-DNA insertion, which was then subjected to sequence analysis.

The sequence flanking the right T-DNA border was subjected to a basicBLASTN search and/or a search of the Arabidopsis Information Resource(TAIR) database (available at the arabidopsis.org website), whichrevealed sequence identity to BAC F22H5, (GI 12331602), mapped tochromosome 1. The junction of the left border of the T-DNA is at nt20167 of F22H5, and the right border junction is at nt 20229. Sequenceanalysis revealed that the T-DNA had inserted in the vicinity (i.e.,within about 10 kb) of the gene whose nucleotide sequence is presentedas SEQ ID NO: 1 and GI 12331602, nucleotides 20955-21335, and which wedesignated PPR2. Specifically, the right border was approximately 500 byupstream of the start codon of SEQ ID NO:1.

Example 3 Analysis of Arabidopsis PPR2 Sequence

The amino acid sequence predicted from the PPR2 nucleic acid sequence ispresented in SEQ ID NO:2 and GI 10092271.

Sequence analyses were performed with BLAST (Altschul et al., 1997, J.Mol. Biol. 215:403-410), PFAM (Bateman et al., 1999), PSORT (Nakai K,and Horton P, 1999, Trends Biochem Sci 24:34-6), and CLUSTALW (ThompsonJ D et al, 1994, Nucleic Acids Res 22:4673-4680), among others.

The PPR2 protein has been characterized as a myb-related protein. PFAManalysis indicated a SANT DNA-binding domain at approximately aminoacids 8-60.

The retroviral oncogene v-myb, and its cellular counterpart c-myb,encode nuclear DNA-binding proteins (Klempnauer and Sippel, 1987, EMBOJ. 6: 2719-2725; Biednkapp et al. 1988, Nature 335: 835-837). Thesebelong to the SANT domain family that specifically recognize thesequence YAAC(G/T)G (Aasland et al. 1996, Trends Biochem. Sci.21:87-88). In myb, one of the most conserved regions consisting of threetandem repeats has been shown to be involved in DNA-binding.

Analysis using BLASTP or TBLASTN identified a number of related proteinsand proteins predicted from nucleic acid (generally EST) sequences inother plant species. Related sequences, which are candidate orthologs,are presented in SEQ ID NOs 3-14 and descriptions from GenBank areprovided below:

SEQ ID NO:3 translation, gi|887283|gb|L38243.1|L38243 BNAF0581E Mustardflower buds Brassica rapa cDNA—ORF 98aa Brassica rapa

SEQ ID NO:4 translation, gi|18459015|gb|BM437293.1|BM437293VVA017C08_(—)54081 An expressed sequence tag database for abiotic st75aa Vitis vinifera

SEQ ID NO:5 translation, gi|15288211|gb|BI472102.11BI472102 sah99e03.ylGm-c1050 Glycine max cDNA clone GENOME SYSTEMS CLONE 97aa Glycine max

SEQ ID NO:6 translation, gi|15258392|gb|B|433702.1|BI433702 EST536463 P.infestans-challenged leaf Solanum tuberosum cDNA clo 88aa Solanumtuberosum

SEQ ID NO: translation, gi|14492357|gb|BI071737.1|BI071737 C063P09UPopulus strain T89 leaves Populus tremula×Populus trem 71aa P o

SEQ ID NO:8 translation, gi|7981380|emb|CAB91874.1| (AJ277944)myb-related protein [Lycopersicon esculentum] 88aa Lycopersiconesculentum

SEQ ID NO:9 gi|5091605|gb|AAD39594.1|AC007858_(—)8 (AC007858) 10A19I.9[Oryza sativa] 126aa Oryza sativa

SEQ ID NO:10 gi|5091604|gb|AAD39593.1|AC007858_(—)7 (AC007858) 10A19I.8[Oryza sativa] 236aa Oryza sativa

SEQ ID NO:11 gi|18394750|ref|NP_(—)564087.1| (NM_(—)101808) myb-relatedprotein, putative [Arabidopsis thaliana] 92aa Arabidopsis thaliana

SEQ ID NO:12 gi|15226604|ref|NP_(—)179759.1| (NM_(—)127736) unknownprotein [Arabidopsis thaliana]·gi|4567225|gb|AAD236 101aa Arabidopsisthaliana

SEQ ID NO:13 gi|15234999|refINP_(—)195636.1| (NM_(—)120086) putativeprotein [Arabidopsis thaliana]·gi|7487341|pir||T08 97aa Arabidopsisthaliana

SEQ ID NO:14 gi|8778436|gb|AAF79444.1|AC025808_(—)26 F18O14.26[Arabidopsis thaliana]

Example 4 Confirmation of Phenotype/Genotype Association

PCR analysis, using primers to sequences in pSKI015 or flanking theinsert, was used to detect lines containing or lacking the insert.W000058335 individuals analyzed in the tertiary screen were genotyped.Results indicated that plants that were homozygous or hemizygous for theinsert were more resistant to P. parasitica infection than plants thatwere homozygous wild-type; 100% of the plants homozygous for the insertand 97% of the plants hemizygous for the insertion received resistancescores of 0 or 1 while only 31% of the wild-type segregants scored 0or 1. These results suggest that the P. parasitica resistance trait inW000058335 is caused by the overexpression of PPR2 and is inherited in adominant manner

RT-PCR analysis showed that the PPR2 gene was overexpressed in plantsfrom the line displaying the P. parasitica resistance phenotype.Specifically, RNA was extracted from tissues derived from plantsexhibiting the resistance phenotype and from wild type COL-0 plants.RT-PCR was performed using primers specific to the sequence presented asSEQ ID NO:1, to other predicted genes in the vicinity of the T-DNAinsertion (At1g75240, At1g75260, and At1g75270), and to a constitutivelyexpressed actin (positive control). The results showed that plantsdisplaying the PPR2 phenotype over-expressed the mRNA for the PPR2 gene,indicating the enhanced expression of the PPR2 gene is correlated withthe PPR2 phenotype.

Example 5 Recapitulation of Pathogen Resistance Phenotype

Arabidopsis plants of the Ws ecotype are transformed by agrobacteriummediated transformation with a construct containing the coding sequencesof the PPR2 gene (At1g75250, alias F22H5.3; G1:10092271) behind theCsVMV promoter and in front of the nos terminator or a control geneunrelated to pathogen resistance. Both of these constructs contain thenptII gene to confer kanamycin resistance in plants. T1 seed isharvested from the transformed plants and transformants selected bygerminating seed on agar medium containing kanamycin. Kanamycinresistant transformants are transplanted to soil after 7 days and grownfor 4 weeks. Control plants are germinated on agar medium withoutkanamycin, transplanted to soil after 7 days and grown in soil for 4weeks

To evaluate pathogen resistance, transformants and control plants aresprayed with a suspension of 1×10⁵ conidia per ml of P. parasitica,incubated at 100% humidity for 1 day, and grown for 6 more days in thegrowth room After this growth period, plants are rated for severity ofdisease symptoms. A score of 0 means the leaves have 0-10% of the numberof conidiophores growing on the leaf surface as a fully susceptibleplant, 1 means 10-25% the number of conidiophores, 2 means 25-50%, 3means 50-75% and 4 means 75-100%. Plants transformed with PPR2, andplants transformed with the control gene are examined.

Degree-of-infection scores are obtained from each plant tested. As agroup, the PPR2 transformants are more resistant to P. parasiticainfection than control plants demonstrating that plants over-expressingPPR2 are significantly more resistant to P. parasitica infection thanwild-type plants.

1. A method of producing a plant having increased pathogen resistance comprising identifying a plant that has an allele in a nucleotide sequence that encodes a Myb polypeptide that confers Peronospora parasitica resistance (PPR2) and that results in increased pathogen resistance compared to plants lacking the allele; and generating progeny of the identified plant, wherein the Myb polypeptide is selected from the group consisting of: the polypeptide sequence of SEQ ID NO: 2; a polypeptide sequence having at least 95% sequence identity to SEQ ID NO: 2; and an Arabidopsis thaliana polypeptide having at least 85% sequence identity to SEQ ID NO: 2, and wherein the generated progeny inherit the allele and have the increased pathogen resistance.
 2. The method of claim 1 that employs candidate gene/Quantitative Trait Locus (QTL) methodology.
 3. The method of claim 1 that employs targeted induced local lesions in genomes (TILLING) methodology.
 4. The method of claim 1, wherein the allele alters the expression of the nucleotide sequence.
 5. The method of claim 1, wherein the allele alters the activity of the Myb polypeptide encoded by the nucleotide sequence.
 6. A plant generated by the method of claim
 1. 7. A plant part obtained from the plant of claim 6, wherein the plant part comprises the allele.
 8. The plant part of claim 7, which plant part is a seed, embryo, meristemic region, callus tissue, leaf, root, shoot, gametophyte, sporophyte, pollen, or microspore.
 9. The plant of claim 6, wherein the nucleotide sequence encodes an Arabidopsis thaliana polypeptide having at least 85% sequence identity to SEQ ID NO:
 2. 10. The plant of claim 9, wherein the nucleotide sequence encodes an Arabidopsis thaliana polypeptide having at least 90% sequence identity to SEQ ID NO:
 2. 11. The plant of claim 10, wherein the nucleotide sequence encodes an Arabidopsis thaliana polypeptide having at least 95% sequence identity to SEQ ID NO:
 2. 12. The transformed plant of claim 11, wherein the nucleotide sequence encodes SEQ ID NO:
 2. 13. The transformed plant of claim 12, wherein the nucleotide sequence comprises SEQ ID NO:
 1. 